Accurate long-read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

To halt North Sea ecosystem degradation, accurate and intensive monitoring of the North Sea ecosystem and its fish is vital to correctly inform management decisions. DNA based techniques and especially the use of environmental (e)DNA from seawater can become a powerful monitoring tool. However, current eDNA based metabarcoding approaches are based on genetic target regions of <500 nucleotides which offers only limited taxonomic resolution.We tested sensitivity and applicability for field samples of newly designed universal fish primer  targeting a 2kb region covering mitochondrial 12S and 16S genes in eDNA samples. Samples were processed using long read nanopore sequencing in combination with the consensus builder Decona and retrieved accurate read identities of up to 99.9%. To test accuracy of the primer, eDNA  was analyzed from a tropical aquarium with a known species composition of bony fish and elasmobranchs. This showed that over 50% of species present can be identified. The majority of remaining reads are identified as -in aquarium present- genera and can be explained by an incomplete reference database for the fish present in the aquarium.  Primers were also applied in North sea eDNA field samples. Distinct species compositions between different locations could be observed and consisted of ecological relevant species and shows the applicability for long-read eDNA metabarcoding in field studies.Incomplete reference databases currently form the main bottleneck to further develop high resolution nanopore based long read sequencing as metabarcoding strategy. Nevertheless, this study shows that long read nanopore sequencing of eDNA can be used to obtain accurate information on the fish and elasmobranch species composition in the North Sea and beyond

Decona: From demultiplexing to consensus for Nanopore amplicon data

Sequencing of long amplicons is one of the major benefits of Nanopore technologies, as it allows for reads much longer than Illumina. One of the major challenges for the analysis of these long Nanopore reads is the relatively high error rate. Sequencing errors are generally corrected by consensus generation and polishing. This is still a challenge for mixed samples such as metabarcoding environmental DNA, bulk DNA, mixed amplicon PCR’s and contaminated samples because sequence data would have to be clustered before consensus generation.To this end, we developed Decona (https://github.com/Saskia-Oosterbroek/decona), a command line tool that creates consensus sequences from mixed (metabarcoding) samples using a single command. Decona uses the CD-hit algorithm to cluster reads after demultiplexing (qcat) and filtering (NanoFilt). The sequences in each cluster are subsequently aligned (Minimap2), consensus sequences are generated (Racon) and finally polished (Medaka). Variant calling of the clusters (Medaka) is optional. With the integration of the BLAST+ application Decona does not only generate consensus sequences but also produces BLAST output if desired. The program can be used on a laptop computer making it suitable for use under field conditions.Amplicon data ranging from 300-7500 nucleotides was successfully processed by Decona, creating consensus sequences reaching over 99,9% read identity. This included fish datasets (environmental DNA from filtered water) from a curated aquarium, vertebrate datasets that were contaminated with human sequences and separating sponge sequences from their countless microbial symbionts.Decona considerably simplifies and speeds up post sequencing processes, providing consensus sequences and BLAST output through a single command. Classifying consensus sequences instead of raw sequences improves classification accuracy and drastically decreases the amount of sequences that need to be classified. Overall it is a user friendly option for researchers with limited knowledge of script based data processing

FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development

The forkhead transcription factor FoxO6 is prominently expressed during development of the murine neocortex. However, its function in cortical development is as yet unknown. We now demonstrate that cortical development is altered in FoxO6+/- and FoxO6-/- mice, showing migrating neurons halted in the intermediate zone. Using a FoxO6-directed siRNA approach, we substantiate the requirement of FoxO6 for a correct radial migration in the developing neocortex. Subsequent genome-wide transcriptome analysis reveals altered expression of genes involved in cell adhesion, axon guidance, and gliogenesis upon silencing of FoxO6. We then show that FoxO6 binds to DAF-16-binding elements in the Plexin A4 (Plxna4) promoter region and affects Plxna4 expression. Finally, ectopic Plxna4 expression restores radial migration in FoxO6+/- and siRNA-mediated knockdown models. In conclusion, the presented data provide insights into the molecular mechanisms whereby transcriptional programs drive cortical development